The present invention relates to methods, agents and compositions for treating plaque-forming diseases, including, but not limited to, Alzheimer""s disease. More particularly, these methods involve the use of (i) plaque derived antigens cloned and displayed on the surface of a display vehicle for in vivo elicitation of antibodies capable of preventing plaque formnation and of disaggregating existing plaques; and (ii) antibodies raised against plaque derived antigens, cloned and displayed on a display vehicle, and which are capable of preventing plaque formation and of disaggregating existing plaques. The present invention further relates to a method of targeting a display vehicle to the brain of an animal, including man.
Alzheimer""s Diseasexe2x80x94Clinical Overview:
Alzheimer""s disease (AD) is a progressive disease resulting in senile dementia. Broadly speaking, the disease falls into two categories: late onset, which occurs in old age (typically above 65 years) and early onset, which develops well before the senile period, e.g., between 35 and 60 years. In both types. of the disease, the pathology is similar, but the abnormalities tend to be more severe and widespread in cases beginning at an earlier age. The disease is characterized by two types of lesions in the brain, senile plaques and neurofibrillary tangles. Senile plaques are areas of disorganized neutrophils up to 150 mm across with extracellular amyloid deposits at the center, visible by microscopic analysis of sections of brain tissue. Neurofibrillary tangles are intracellular deposits of tau protein consisting of two filaments twisted about each other in pairs.
Senile Plaques and Other Amyloid Plaques:
The principal constituent of the senile plaques is a peptide termed Axcex2 or beta-amyloid peptide (xcex2AP). The amyloid beta peptide is an internal fragment of 39-43 amino acids of a precursor protein termed amyloid precursor protein (APP). Several mutations within the APP protein have been correlated with the presence of Alzheimer""s disease (See, e.g., Goate et al., Nature 349,704, 1991, valine717 to isoleucine; Chartier Harlan et al. Nature 353, 844, 1991, valine717 to glycine; Murrell et al., Science 254, 97, 1991, valine717 to phenylalanine; Mullan et al., Nature Genet. 1, 345, 1992, a double mutation changing lysine595-methionine596 to asparagine595-leucine596).
Such mutations are thought to cause Alzheimer""s disease by increased or altered processing of APP to beta-amyloid, particularly processing of APP to increased amounts of the long form of beta-amyloid (i.e., Axcex21-42 and Axcex21-43). Mutations in other genes, such as the presenilin genes, PS1 and PS2, are thought indirectly to affect processing of APP to generate increased amounts of long form beta-amyloid (see Hardy, TINS 20, 154, 1997). These observations indicate that beta-amyloid, and particularly its long form, is a causative element in Alzheimer""s disease.
Amyloid deposits comprise a peptide aggregated to an insoluble mass. The nature of the peptide varies in different diseases but in most cases, the aggregate has a beta-pleated sheet structure and stains with Congo Red dye. In addition to Alzheimer""s disease (AD), both late and early onset, other diseases characterized by amyloid deposits are, for example, SAA amyloidosis, hereditary Icelandic syndrome, multiple myeloma, and spongiform encephalopathies, including mad cow disease, Creutzfeldt Jakob disease, sheep scrapie, and mink spongiform encephalopathy (see, for example, Weissmann et al., Curr. Opin. Neurobiol. 7, 695-700, 1997; Smits et al., Veterinary Quarterly 19, 101-105, 1997; Nathanson et al., Am. J. Epidemiol. 145, 959-969, 1997).
The peptides forming the aggregates in these other diseases are serum amyloid A, cystantin C and IgG kappa light chain, respectively, for the first three, and prion protein for the others.
Other peptides or proteins with evidence of self aggregation are also known, such as, but not limited to, amylin (Young A A. et al., 1994, FEBS Lett, 343(3);237-41); bombesin, caerulein, cholecystokinin octapeptide, eledoisin, gastrin-related pentapeptide, gastrin tetrapeptide, somatostatin (reduced), substance P; and peptide, luteinizing hormone releasing hormone, somatostatin N-Tyr (Banks and Kastin, Prog Brain Res., 91:139-4, 1992).
Treatment:
U.S. Pat. No. 5,688,561 to Solomon teaches methods of identifying monoclonal antibodies effective in disaggregating protein aggregates and preventing aggregation of such proteins. Specifically, U.S. Pat. No. 5,688,561 demonstrates anti-beta-amyloid monoclonal antibodies effective in disaggregating beta-amyloid plaques and preventing beta-amyloid plaque formation in vitro. U.S. Pat. No. 5,688,561 stipulates the in vivo use of such antibodies to prevent plaque formation by aggregation of beta-amyloid or to disaggregate beta-amyloid plaques which have already formed. These teachings do not, however, identify an epitope to be employed to generate such antibodies. In addition, these teachings do not provide means with which to enable the penetration of such antibodies into the brain through the blood brain barrier (BBB). Furthermore, this patent fails to teach the use of phage display technology as a delivery method for antigens or antibodies. Yet furthermore. no experimental results demonstrating the in vivo effectiveness of such antibodies are demonstrated by U.S. Pat. No. 5,688,561.
EP 526511 by McMichael teaches administration of homeopathic dosages (less than or equal to 10xe2x88x922 mg/day) of beta-amyloid to patients with pre-established AD. In a typical human with about 5 liters of plasma, even the upper limit of this dosage would be expected to generate a concentration of no more than 2 pg/ml. The normal concentration of beta-amyloid in human plasma is typically in the range of 50-200 pg/ml (Seubert et al., Nature 359, 325-327 1992). Because this proposed dosage would barely alter the level of endogenous circulating beta-amyloid and because EP 526511 does not recommend the use of an adjuvant, it seems implausible that any therapeutic benefit would result therefrom.
PCT/US98/25386 by Schenk and a Nature paper by Schenk et al. (Nature, 400:173-177, 1999) teach administration of beta-amyloid immunogens to a patient in order to generate antibodies to prevent formation of plaques or dissolve existing plaques. According to Schenk, 50 to 100 mg of antigen are required, 1 to 10 mg if an adjuvant is employed. These teachings also stipulate that a similar effect may be achieved by direct administration of antibodies against beta-amyloid, in both cases disregarding the blood brain barrier which, under normal circumstances, prevents the penetration of antibodies into the brain.
It is also important to note that these teachings are typically restricted to the use of xe2x80x9c . . . any of the naturally occurring forms of beta-amyloid peptide, and particularly the human forms (i.e., Axcex239, Axcex240, Axcex241, Axcex242 or Axcex243)xe2x80x9d or xe2x80x9c . . . longer polypeptides that include, for example, a beta-amyloid peptide, active fragment or analog together with other amino acidsxe2x80x9d, or xe2x80x9cmultimers of monomeric immunogenic agentsxe2x80x9d.
These teachings ignore, however, earlier data teaching that the first 28 amino acids of beta-amyloid are sufficient to elicit antibodies which both disaggregate and inhibit aggregation of beta-amyloid plaques in vitro (Hanan and Solomon, Amyloid: Int. J. Exp. Clin. Invest. 3:130-133, 1996; Solomon et al., Proc. Natl. Acad. Sci. U.S.A. 93:452-455, 1996; Solomon et al., Proc. Natl. Acad. Sci. U.S.A. 94:4109-4112, 1997).
Schenk and Schenk et al. both fail to teach the use of the N-terminal epitope of beta-amyloid plaques which is known to be a sequential epitope composed of only four amino acid residues (EFRH, SEQ ID NO:1) located at positions 3-6 of the beta-amyloid peptide (Frenkel D., J. Neuroimmunol., 88:85-90,1998). Antibodies against this epitope have subsequently been shown to disaggregate beta-amyloid fibrils, restore beta-amyloid plaques solubilization and prevent neurotoxic effects on PC 12 cells (Solomon, B. et al., Proc. Natl. Acad. Sci. USA. 94:4109-4112, 1997; and Solomon, B., et al., Proc. Natl. Acad. Sci. USA., 93:452-455,1996).
This epitope has been independently confirmed as the epitope bound by anti-aggregating antibodies using random combinatorial hexapeptide phage display (Frenkel and Solomon, J. of Neuroimmunol. 88:85-90, 1998).
The EFRH (SEQ ID NO:1) epitope is available for antibody binding when beta-amyloid peptide is either in solution or in aggregates. Blocking of this epitope by a monoclonal antibody prevents self-aggregation and enables resolubilization of already formed aggregates.
These findings suggest that the teachings of Schenk and colleagues are inefficient at best. Since, as has already been mentioned hereinabove, the normal concentration of beta-amyloid in human serum is 50-200 pg/ml, immunization with that peptide could be expected to produce either low antibody titers or high toxicity if strong adjuvants are used and as such it is not applicable for therapy. Indeed, in order to achieve significant serum titers of antibody against beta-amyloid a series of 11 monthly injections was required (Schenk et al., Nature, 400:173-177, 1999). The degree to which these serum titers will persist over time is not yet known, and this point is especially crucial with respect to early onset Alzheimer""s disease.
Schenk and colleagues further teach that an immunogenic peptide such as beta-amyloid may be displayed upon the surface of a virus or bacteria. However, they fail to teach use of an antigen so displayed to effect immunization. No mention is made of defining an epitope in this context and no experimental data is provided either. In addition, delivery of antibody displayed on a display vehicle is not taught by Schenk or Schenk et al. altogether.
Collectively, the prior art fails to teach means with which an effective titer of anti-aggregation antibodies can be generated in vivo in a short time and/or be introduced into the brains of patients suffering a plaque-forming diseases. In addition, the persistence of titers generated via prior art teachings has not been established.
There is thus a widely recognized need for, and it would be highly advantageous to have, effective means of disaggregating amyloid plaques in vivo which would have lasting effect, high efficiency, rapid onset, no adverse effect on the treated subject and which is readily amenable to large scale production.
According to one aspect of the present invention there is provided a method of treating a plaque forming disease comprising the steps of (a) displaying a polypeptide on a display vehicle, the polypeptide representing at least one epitope of an aggregating protein associated plaque formation in the plaque forming disease, the at least one epitope being capable of eliciting antibodies capable of disaggregating the aggregating protein and/or of preventing aggregation of the aggregating protein; and (b) introducing the display vehicle into a body of a recipient so as to elicit the antibodies capable of disaggregating the aggregating protein and/or of preventing aggregation of the aggregating protein.
According to another aspect of the present invention there is provided an agent for treating a plaque forming disease comprising a displayvehicle displaying a polypeptide representing at least one epitope of an aggregating protein associated with plaque formation in the plaque forming disease, the at least one epitope being capable of eliciting antibodies capable of disaggregating the aggregating protein and/or of preventing aggregation of the aggregating protein.
According to yet another aspect of the present invention there is provided a pharmaceutical composition for treating a plaque forming disease comprising an effective amount of a display vehicle displaying a polypeptide, the polypeptide representing at least one epitope of an aggregating protein associated with plaque formation in the plaque forming disease, the at least one epitope being capable of eliciting an effective amount of antibodies capable of disaggregating the aggregating protein and/or of preventing aggregation of the aggregating protein, the pharmaceutical composition further comprising a pharmaceutically acceptable carrier.
According to still another aspect of the present invention there is provided a method of preparing a display vehicle for treating a plaque forming disease, the method comprising the step of genetically modifying a genome of a display vehicle by inserting therein a polynucleotide sequence encoding a polypeptide representing at least one epitope of an aggregating protein associated with plaque formation in the plaque forming disease, the at least one epitope being capable of eliciting antibodies capable of disaggregating the aggregating protein and/or of preventing aggregation of the aggregating protein, such that when the display vehicle propagates the polypeptide is displayed by the display vehicle.
According to an additional aspect of the present invention there is provided a method of treating a plaque forming disease comprising the steps of (a) displaying a polypeptide representing at least an immunological portion of an antibody being for binding at least one epitope of an aggregating protein associated with plaque formation in the plaque forming disease, the binding capable of disaggregating the aggregating protein and/or of preventing aggregation of the aggregating protein; and (b) introducing the display vehicle into a body of a recipient so as to disaggregate the aggregating protein and/or prevent its aggregation.
According to still an additional aspect of the present invention there is provided a method of introducing a display vehicle lacking an engineered targeting moiety into a brain of a recipient, the method comprising the step of administering the display vehicle intranasally to the recipient.
According to further features in preferred embodiments of the invention described below, the step of introducing the display vehicle into the body of the recipient so as to disaggregate the aggregating protein is effected through an olfactory system of the recipient.
According to yet another additional aspect of the present invention there is provided an agent for treating a plaque forming disease comprising a display vehicle displaying a polypeptide representing at least an immunological portion of an antibody which can bind at least one epitope of an aggregating protein associated with plaque formation in the plaque forming disease, the immunological portion of the antibody being capable of disaggregating said aggregating protein and/or of preventing aggregation of the aggregating protein.
According to yet an additional aspect of the present invention there is provided a pharmaceutical composition for treating a plaque forming disease comprising an effective amount of a display vehicle displaying a polypeptide representing at least an immunological portion of an antibody which can bind at least one epitope of an aggregating protein associated with plaque formation in the plaque forming disease, the immunological portion of the antibody being capable of disaggregating the aggregating protein and/or of preventing aggregation of the aggregating protein, the pharmaceutical composition further comprising a pharmaceutically acceptable carrier.
According to still an additional aspect of the present invention there is provided a method of preparing a display vehicle for treating a plaque forming disease comprising the step of genetically modifying a genome of a display vehicle by inserting therein a polynucleotide sequence encoding at least an immunological portion of an antibody capable of binding at least one epitope of an aggregating protein associated with plaque formation in the plaque forming disease, the immunological portion of the antibody being capable of disaggregating the aggregating protein and/or of preventing aggregation of the aggregating protein.
According to further features in preferred embodiments of the invention described below, the plaque forming disease is selected from the group consisting of early onset Alzheimer""s disease, late onset Alzheimer""s disease, presymptomatic Alzheimer""s disease, SAA amyloidosis, hereditary Icelandic syndrome, senility, multiple myeloma and a spongiform encephalopathy.
According to still further features in the described preferred embodiments, the aggregating protein is selected from the group consisting of beta-amyloid, serum amyloid A, cystantin C, IgG kappa light chain and prion protein.
According to additional further features in preferred embodiments of the invention, the display vehicle is selected from the group consisting of a double stranded DNA virus, a single stranded DNA virus, an RNA virus, a bacteria and a prion.
According to still further features in the described preferred embodiments, the display vehicle is a virus.
According to still further features in the described preferred embodiments, the display vehicle is a bacteriophage.
According to still further features in the described preferred embodiments, the display vehicle is a filamentous bacteriophage.
According to further features in described preferred of the invention, the bacteriophage display vehicle is capable of propagating within bacterial flora of the host.
According to further features in described preferred embodiments of the invention, the bacteriophage display vehicle is capable of propagating within E. coli. 
According to still further features in the described preferred embodiments the bacteriophage display vehicle is fd.
According to further features in described preferred embodiments of the invention, the display vehicle is incapable of propagation in vivo.
According to still further features in the described preferred embodiments, a triple dose of 1010 units of the chosen display vehicle induces an antibody titer of at least 1:50,000 within 30 days of administration, as measured by ELISA. The present invention successfully addresses the shortcomings of the presently known configurations by providing methods, agents, and pharmaceutical compositions for quickly and reliably preventing or reversing the progression of a plaque forming disease. The invention further includes methods for preparing display vehicles for use as agents, or as part of pharmaceutical compositions, associated with prevention or reversal of plaque forming diseases.